His tag - protein purification

Hi,I am expressing HIV gp41 protein cloned in pET32a+ vector. After expression , I like to purify it with HIS tag method. I am trying with Ni column but 70% of the protein comes in the flowthrough. Could you please give me a protocol to avoid this.sincerelyB.Veera.

We too have had problems purifying His-tagged gp41 in its native confirmation. However, under denaturing conditions (8M urea), 95% of gp41 was able to bind Ni columns. Therefore, one can conclude that the his-tag was buried in the 3D confirmation of the native protein. If you need to purify "native" gp41, try moving the his-tag to the other end of the protein and see if that works.